We are addressing two key issues and associated hypotheses related to epigenetic and signal-transduction mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. The first key issue is to evaluate the chromatin status of the OT &VP genes in the SON. Here the hypothesis is that certain chromatin modifications in the two genes are specifically expressed in the OT and VP neuronal phenotypes .More specifically, that the cell specific expression of OT and VP in the SON is due to the acetylation and methylation of histones on the OT gene in VP cells &the VP gene in OT cells. This type of chromatin modification is very dynamic &therefore reversible, so coexpression of OT &VP in the SON could occur under some circumstances (see Glasgow et al, 1999;Xi et al, 1999.) To address this hypothesis, we are now doing epigenetic studies by:1) analysing histone acetylation &methylation patterns in OT &VP chromatin in SON and in other specific genes, under various physiological conditions in vivo, and 2) to test effects of acetylation/deacetylation inhibitors on OT &VP chromatin and hnRNA transcription in vivo. An additional hypothesis is that the cell-specific expression of the OT &VP genes in the HNS and the silencing of these genes in most brain areas such as in cortex, striatum, cerebellum, etc, and possibly also for OT in the SCN, is due to the DNA methylation of these genes in the non-expressing cells (e.g, as in imprinting). The stable silencing of the OT &VP genes in areas such as cerebral cortex, etc, and possibly of OT in the SCN could be due to DNA methylation, which is much less dynamic and in many cases irreversible (e.g, in imprinting). Experiments are under consideration to test this hypothesis. The second key question is to determine which neural signals, signal transduction, and transcriptional mechanisms regulate OT and VP gene expression in the SON. Previous studies from our laboratory showed that stereotaxic Alzet miniosmotic pump delivery of tetrodotoxin (TTX) blocks acute systemic hyperosmotic induction of immunoreactive c-fos and VP hnRNA in the SON (Shahar et al, 2004, Kawasaki et al, 2009) which suggests that synaptic input is an essential signal for osmotic regulation of VP gene expression. Acute systemic hyperosmotic stimuli increases VP but not OT hnRNA in the SON , although both peptides equally increase in secretion from the neural lobe (Yue et al, 2008a;2008b) further suggesting a profound difference in excitation-secretion vs excitation-transcription coupling mechanisms in the OT vs VP phenotypes. In addition, while direct neurotransmitter stimulation is necessary it may not be sufficient to regulate gene expression. Recent studies by Kawasaki et. al (2009) showed that neither glutamate, acting via NMDA and AMPA receptors. known to be critical in increasing VP secretion by the HNS during acute hyperosmotic stimulation, nor direct osmotic stimulation of the MCNs are able by themselves to increase VP gene expression in the SON. Given these data we hypothesize that The signal transduction cascade involved in acute osmotic evoked stimulation of VP gene expression in the SON involves activation of specific calcium and/or cAMP-dependent intracellular kinases and/or phosphatases acting ultimately on CREB and related transcription factors. Related experiments in progress are to: 1) Evaluate effects of direct application of putative second messengers (e.g, calcium ion &cAMP) on VP transcription &2) effects of various kinase inhibitors and kinase siRNAs on the acute osmotic stimulation of VP transcription.